This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of our proposed is to determine the structural basis for 3? splice site recognition. The majority of human gene transcripts are regulated by pre-mRNA splicing. The splice sites are sequentially recognized by protein and RNA components of the spliceosome, a pre-mRNA splicing machine composed of more than 100 proteins and 5 small nuclear RNAs. A complex composed of essential splicing factors SF1, U2AF65, and U2AF35 recognizes the pre-mRNA signals and recruits the core splicing machinery to a target splice site. Specific aims of this proposal address the following central questions concerning the critical early stages of pre-mRNA splicing: (1) What is the structure of a highly-conserved SF1 domain that lacks structural homologues? (2) By what structural means does phosphorylation of this SF1 domain enhance association with U2AF? (3) By what structural means does U2AF adapt to diverse splice sites? (4) What is the three-dimensional architecture of the SF1 / U2AF complex with the target splice site? Synchrotron radiation is essential to address the aims of this proposal for reasons including: (i) Tunable wavelengths are necessary for multiwavelength anomalous dispersion experiments;(ii) The robotic capability greatly facilitates the extensive screening required to identify useful crystals for Aim 1;(iii) Crystal size is small and consequently diffraction is prohibitively weak using conventional in-house x-ray sources.